Peptide from soluble form of acetylcholinesterase, active as a calcium channel modulator

ABSTRACT

The acetylcholinesterase 14-mer peptide AEFHRWSSYMVHWK acts alone or in synergism with  2 -amyloid to contribute to neuronal degeneration, e.g. in Parkinson&#39;s and Alzheimer&#39;s diseases, perhaps by exerting calcium channel opening activity. Antibodies and other compounds which inhibit the biological activity are useful for prophylaxis or treatment.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention concerns the enzyme acetylcholinesterase (AChE) in whichthe inventors have identified a biologically active peptide.

2. Description of the Related

The classical or cholinergic role of AChE is to degrade enzymaticallyextracellular acetylcholine. However, it has long been known that AChEexists also in a soluble form, (not a requirement for its classicenzymatic role) and is found in parts of the body where there is littleor no acetylcholine. It is becoming widely accepted that AChE has anon-cholinergic function, though the biochemical basis for this functionremains unclear.

It is believed that excessive AChE may enhance calcium entry into cellsindependent of its normal enzymatic action. Elevated cellular calciumlevels may lead to a range of pernicious consequences, includingundesirable changes in gene expression and, more importantly,mitochondrial swelling which may thereby compromise ATP metabolism andmay indeed lead to apoptosis or programmed cell-death. Disease stateswhich may be implicated include Parkinson's disease, Alzheimer'sdisease, stroke and malignancy.

The cytoplasm of cells typically contains calcium at concentrations ofthe order of 1 μm. Calcium is present intracellularly in the endoplasmicreticulum in millimolar concentrations. Extracellular body fluidscontain calcium also in millimolar concentrations. A calcium pumpoperates to maintain this substantial concentration difference betweenthe cytoplasm and the endoplasmic reticulum, and thapsigargin is knownto be implicated in the breakdown of this pump. Similarly a calcium pumpnormally functions between the cytoplasm and the extracellular fluid. Itis believed that the consequences of the action of excessive AChE may becomparable to the breakdown of these pumps.

AChE, acting in a non-cholinergic capacity, has been shown to play animportant part in the normal and abnormal functioning of the substantianigra, the region affected in Parkinson's disease. There are are threepossible ways in which AChE may have toxic effects:

-   (i) excessive AChE may be released as a consequence of compensatory    mechanisms known to occur in that disorder;-   (ii) excessive glutamatergic activity known to occur in Parkinson's    disease may lead to over-stimulation of calcium channel    N-methyl-D-aspartate (NMDA) glutamate receptors, thereby converting    a physiological situation to a pathological one;-   (iii) normal levels of AChE may act synergistically with fragments    of β-amyloid precursor proteins known to be present in the    Parkinsonian substantia nigra.

AChE, again acting in a non-cholinergic capacity, may be an importantcontributing factor in Alzheimer's disease. In transgenic mice withexcessive AChE there are cognitive deficits reminiscent of Alzheimer'sdisease. Moreover Alzheimer's disease has been directly associated withinappropriate levels and forms of AChE. Excessive AChE may act toenhance calcium entry through overactivation of otherwise normaladaptive processes via a mechanism discussed in the experimental sectionbelow.

Current therapies for both degenerative diseases are somewhatinadequate. Anti-Parkinsonian drugs which target dopamine substitutiondo not arrest neuronal cell loss, and newer drugs aiming to blockcalcium entry directly may have poor net payoff in terms of neuronalhealth and in addition would have widespread undesirable effects in boththe central nervous system and peripheral tissue. Moreover, drugs usedin Alzheimer's disease which exclusively target the cholinergic system,neglect areas where AChE may be having its pivotal non-cholinergicfunction. Previous attempts to target calcium channel activity intherapy for neurodegenerative disorders have been hampered by thenon-selective effects of the compounds available.

In order to be conveniently administered, a compound for treatment ofdisorders of the central nervous system, or more particularly of thebrain, needs to be capable of crossing the blood-brain barrier. AChE isnot capable of doing this, though a small lipid-soluble analogue of partof this molecule might be. Workers in the field have been seekingbiologically active peptides based on the AChE molecule for more thanten years, in the hope of thereby achieving a more effective andselective treatment for disorders of the central nervous system such asAlzheimer's and Parkinson's diseases.

It is known that antagonism of NMDA receptors is being explored as atherapy for stroke. The present invention is expected to findapplication in specific therapies for combating stroke and otherproblems of cerebral circulation.

Abnormal cholinesterase expression occurs in several types of tumourcells. Although the role of cholinesterases in tumorigenesis is unclear,the fact that AChE and BuChE (butyryl cholinesterase) may be involved inthe control of cell growth and proliferation during early developmentsuggests that the amplification of cholinesterase genes may influencethe ability of tumour cells to proliferate more rapidly. According tothe invention, antagonists of the non-cholinergic action of AChE areexpected to be of interest in the prophylaxis and treatment of cancer.

Several separate lines of evidence suggest that motor neurones mayshare, along with the neurones that are lost in Parkinson's disease(substantial nigra) and in Alzheimer's disease (basal forebrain, locuscouruleus, raphe nucleus) several distinctive features as well as thecommon characteristic of releasing AChE in a non-cholinergic capacity.The released AChE may have a novel action, as in the regions prone toParkinsonian or Alzheimer degeneration to enhance developmentalmechanisms in immature populations of motor neurones but exert toxicactions if inappropriately reactivated in mature systems. TheAChE-peptide described herein may also therefore be pivotal in theaetiology of Motor Neurone Disease. The undisclosed finding supportingthis claim is that in pilot studies, the AChE peptide binds bilaterallyto selective sites within the spinal cord.

Amyloid precursor protein (APP) is known to have similar features toAChE as follows. Both AChE and APP are secreted from neurons into thecerebro spinal fluid (CSF), where for both AChE and APP there is adecrease in CSF levels in Alzheimer's disease. Both AChE and APP canhave trophic functions.

Both AChE and β-amyloid enhance calcium entry through NMDA receptors.Both AChE and APP activate potassium channels, probably linked tochanges in intracellular calcium. Both AChE and β-amyloid activatemacrophages. Low stimulation of NMDA receptors has trophic effectswhereas high stimulation is toxic. The dual trophic-toxic action of bothAPP and AChE may thus be mediated via NMDA receptors. A similar dualaction via NMDA receptors has already been shown for the trophic factorBDNF in cortical cells. Finally, β-amyloid and the monomer of AChE canbind together as a complex.

This invention results from the inventors' identification of a region ofthe AChE molecule from which a biologically active peptide (obtainedeither synthetically or by endogenous processing) can be derived. Thepeptide consists of 14 residues of AChE from residue 535 to residue 548of the mature protein (in the translation of the mRNA sequence, EMBLaccession hsache. empri, number M55040, beginning at nucleotide 310).The sequence of this peptide is amino Ala-Glu-Phe-His-Arg-Trp-Ser-Ser-Tyr-Met-Val-His-Trp-Lys-carboxy (SEQ. ID No: 1), or inthe one letter code, AEFHRWSSYMVHWK. The inventors propose that this, ora related, peptide from this region of AChE acts alone or in synergismwith a fragment of beta-amyloid to contribute to neuronal degeneration.The invention thus provides in one aspect a peptide containing at leastsix amino acid residues and having at least 70% homology with part orall of the above sequence. Preferably the peptide contains at least 12amino acid residues having at least 90% homology with the abovesequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts multiple sequence alignments of five AchE sequences,three BuChE sequences and the human amyloid precursor polypeptide at theregion of interest. Specifically, it depicts an alignment of polypeptidesequences of hum AChE (human AChE; SEQ ID NO: 6), rab AChE (rabbit AChE;SEQ ID NO: 7), mus AChE (mouse AChE; SEQ ID NO: 8), rat AChE (rat AChE;SEQ ID NO: 9), Bov AChE (bovine AChE; SEQ ID NO: 10), hum BuChE (humanBuChE SEQ ID NO: 11), rab BuChE (rabbit BuChE; SEQ ID NO: 12), mus BuChE(mouse BuChE; SEQ ID NO: 13) and hum Amyl (human A4 amyloid precursorpolypeptide: SEQ ID NO: 14). Residues in bold are conserved across allsequences. Boxed residues are shared by all AChEs and hum Amyl but noneof the BuChE sequences. Amyloid peptide residues 1–42 are shown within abox. The bar above the alignment indicates the position of the AChE andBuChE synthetic peptides, while the bar below the alignment identifiesthe synthetic APP peptide.

It appears that the two amino acid residues-Val-His-, appearing atpositions 11 and 12 in the above sequence, may be of criticalimportance. Thus the invention also envisages peptides comprising orconsisting of the four-mer sequence YMVH (SEQ. ID No: 3) or MVHW (SEQ.ID No: 4) or VHWK (SEQ. ID No: 5) and having at least 70% homology withpart or all of the above AChE sequence.

A somewhat similar peptide is present in a region of the β-amyloidprecursor polypeptide. This region lies at the amino terminus of the 42residue peptide that accumulates in Alzheimer's disease and has thesequence amino -Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-carboxy (SEQ. ID No: 2), or in the one lettercode, DAEFRHDSGYEVHHQK, corresponding to residues 597–612 of thetranslation of the human amyloid A4 precursor polypeptide (EMBLaccession hsafpa4. empri number Y00264, beginning at nucleotide 148).

The accompanying table shows the multiple sequence alignment of 5 AChEsequences, three BuChE sequences and the human amyloid precursorpolypeptide at the region of interest. As reported in the experimentalsection below, the human amyloid precursor fragment does not itselfexert calcium channel opening activity, but it does enhance the activityof the AChE fragment. The BuChE fragment appears inactive, both aloneand together with the AChE fragment. In another aspect the inventionthus envisages a mixture of AChE peptide with another peptide having atleast four amino acid residues preferably including VH and having atleast 70% homology with the above β-amyloid precursor sequence.

There are various ways in which this AChE peptide or this peptidemixture may be used:

-   a) Since the AChE peptide is shown to have nanomolar affinity for a    binding site in the vulnerable cells, the peptide (or mixture) can    be labelled with a signal moiety, or alternatively immobilised, and    used to locate and identify the receptor site of the cells. The    nature of this signal moiety is not material, and the technique of    labelling peptides with signal moieties is well known. The peptide    (or mixture) can be used as an affinity ligand for the selective    retrieval of the receptor molecule itself from preparations derived    from those vulnerable cells. Additionally, the peptide affinity    ligand could be used to screen an appropriate cDNA expression    library to isolate a cDNA encoding the binding site directly. Once    that receptor site is known, it will be possible to modify or    control its properties.-   b) An alternative and preferred approach is to find a substance that    inhibits the action of the biologically active peptide or mixture.    For example an antibody or other substance which binds to the    peptide would be expected to inhibit its biological action.    Structural properties of the active peptide itself, together with a    combinatorial analysis of the optimal peptide sequence for    biological activity, will provide additional information. This    structural information may suggest a family of synthetic    (non-peptide) compounds that could rationally be tested for    efficacy.

In a further aspect, the invention thus envisages a compound whichinhibits a biological activity of the AChE peptide or peptide mixturedescribed above. The biological activity may perhaps be modulating,directly or indirectly a calcium-channel-opening activity. The compoundwill preferably be capable of crossing the blood-brain barrier.

Thus the non-cholinergic action of AChE, as mimicked by its 14 residuepeptide, may be selectively blocked by a synthetic compound devised inthis way. Moreover, the process of developing such a synthetic inhibitoris simplified by the demonstration of biological activity in such asmall sub-fragment of the AChE molecule. A consequence should be thatthe synthetic compound offers a more physiological action, thusreduction of calcium entry into vulnerable cells rather than completeabolition. In addition, this action should occur selectively, only inlocations within the brain where AChE has a non-cholinergic action. Itshould be noted that these are the very sites primarily affected by cellloss in Alzheimer's and Parkinson's diseases. Thus use of thesesynthetic compounds should avoid widespread disruption of cellularcalcium regulation, by offering a highly region-selective action withinthe brain.

EXAMPLE 1 Strategy for the Identification of a Receptor for the AChEPeptide

1. Use the peptide, tagged with biotin, to search for a cell type with ahigh affinity binding site for the peptide. Note that this search willbegin with neuronal-derived tissue culture cell lines that should begood candidates. Functional significance for the binding of the AChEpeptide will be assessed by looking for physiological effects of peptidebinding, such as transient calcium currents.

2. Having identified a cell type with a high affinity binding site forthe peptide, the receptor will be identified by ligand overlay blottingand intracellular localisation by indirect detection of the biotinylatedpeptide using a streptavidin conjugated fluorochrome. Subsequently thereceptor will be purified either by affinity chromatography usingimmobilised peptide, or by conventional column chromatography using theability of the peptide to bind to column fractions as an assay to followpurification.

3. The purified receptor will be subject to N-terminal microsequencing(or tryptic fragments will be purified by HPLC and microsequenced if thereceptor molecule as isolated proves to be N-terminally blocked). Thepeptide sequences obtained in this way will be compared with anon-redundant compilation of available peptide sequence databases toidentify any similarities (or identity with a known surface molecule).The sequences will also be compared with expressed sequence tag (EST)databases in case the mRNA for the receptor has already been obtained asa cDNA by chance in a random library construction and sequencingproject.

4. If the strategy in (3) does not identify a cDNA sequence, the peptidesequences will be back-translated to provide nucleotide sequences fromwhich oligonucleotides will be constructed. These oligonucleotides willbe used to amplify regions of the parent mRNA by reverse transcriptionof total cellular RNA (from the cell type used in the originalbiochemical isolation), followed by specific amplification with eachpossible primer pair using the polymerase chain reaction (PCR). ThesePCR products will be directly sequenced by cycle sequencing using anApplied Biosystems automated sequencer.

5. The sequences obtained from these PCR products will be compared withsequences in existing nucleotide databases as in. (3). If thiscomparison reveals that an identical sequence has previously beenobtained, then a strong candidate for the receptor gene is available(Note that this does not imply that the sequence that has been obtainedpreviously has already been implicated in any way in the functions thatthe invention ascribes to this molecule).

6. If no identical or highly similar sequences are identified in (5),then the PCR-derived nucleotide fragments will be used as radiolabelledprobes to screen a cDNA library constructed by oligo-dT primed reversetranscription from the mRNA of the cell type used in the originalbiochemical isolation. This will identify candidate cDNA clones whichwill then be sequenced as above. The identity of the candidate cDNAswith the protein of interest will be confirmed initially bydemonstrating that the clone contains the sequences of the otherPCR-derived nucleotide fragments. If an incomplete cDNA clone isobtained then 5′ extension will be carried out using the RACE technique(Rapid amplification of cDNA ends).

7. The function of the protein encoded by the cDNA will be confirmed byexpression of the full-length protein using a transient eukaryoticexpression vector in cells previously shown not to have a high affinitybinding site for the AChE peptide. Expression of the protein in thesecells should result in the appearance of a high affinity binding sitefor the AChE peptide on these transfected cells. This will confirm thatthe correct sequence has been identified.

8. The cDNA sequence will be used to express the protein in abaculovirus-infected insect cell system in order to obtain large amountsof pure protein for structural studies. Note that this may require theconstruction of a soluble ectodomain fragment if the protein is atransmembrane molecule. The structural studies will include circulardichroism (CD) measurements, 2-D nuclear magnetic resonance analysis(NMR) and attempts at crystallisation. Pure receptor protein will alsofacilitate detailed analysis of the binding of peptides, or candidatenon-peptide agonists (or antagonists), to the receptor using surfaceplasmon resonance methods.

9. Once the AChE ligand and its receptor are known, there are variousways of controlling or preventing their action:

-   a) Destroy the ligand, i.e. identify a protease that cleaves the    active peptide ligand into an inactive form and promote its activity    e.g. by inducing it or impregnating it.-   b) Prevent production of the ligand, i.e. identify a protease that    produces the ligand and inhibit that.-   c) Sequester the ligand and remove it with antibody or with soluble    receptor ectodomain. A modification of this approach is possible if    the AChE/β-amyloid synergy results from competition for a high    affinity sequestration site different from that which produces the    biological effect. Introduction of excess of high affinity site will    reduce the biological effect.-   d) Block the receptor with an antagonist, e.g. design an analogue    that binds the receptor, competes with endogenous peptide, but does    not raise calcium levels. In an experimental system showing binding    and calcium ion signals, it will be possible to assay for a class of    compounds that bind the receptor, compete with the peptide ligand,    but do not themselves activate the receptor.-   e) Uncouple the receptor from the cellular response, e.g. by    preventing ligand binding to the receptor from causing cellular    response by blocking a second messenger that is preferably unique to    the system.

EXAMPLE 2

The 14-mer AChE peptide, the corresponding 14-mer human BuChE peptide(AGFHRWNNYMMDWK) (SEQ. ID No: 15)and the 16-mer β-amyloid peptide weresynthesised and used in studies to assess their biological activity,with the summarised results given in Examples 3 to 7

EXAMPLE 3

(i) Electrophysiological Studies

These experiments are performed on slices of guinea-pig midbrainmaintained in vitro. Intracellular recordings are made from a rostralpopulation of neurons in the substantia nigra under current clampconditions. All results listed below are obtained in the presence of thesodium channel blocker tetrodotoxin. In order to facilitatevisualisation of calcium-mediated potentials triggered by activation ofNMDA receptors, all experiments are performed in magnesium-freeperfusate. Under these conditions, results to date indicate:

-   (a) In 11 neurons, in concentrations ranging from 10⁻⁷M to 10⁻⁶M,    the peptide fragment derived from AChE has a selective and    reversible action reminiscent of the actions of AChE itself, i.e.    with lower doses/less sensitive situations there is an enhanced    calcium influx. This effect is followed by, in sustained    applications/stronger doses/more sensitive neurons, a marked    reduction in the calcium potentials.-   (b) Under conditions where AChE is normally effective and under    magnesium-free conditions, the comparable BuChE fragment appears    without corresponding effect (n=3), and the analogous fragment of    β-amyloid also appears ineffective (n=4). However, a synergism    between the peptide derived from AChE and this fragment of β-amyloid    is reflected in a reduction in the evoked calcium potential (n=7)    followed by the generation of large spontaneous    thapsigargin-sensitive calcium currents oscillating in a biphasic    manner (n=3).-   (c) In 6 neurons, application of NMDA, which on its own produces a    ‘physiological’ depolarisation, results, under identical conditions,    in severe metabolic stress of the cell after treatment with the AChE    peptide at a concentration as low as 10⁻⁷M, or at an even lower    concentration when combined with the amyloid peptide.

High doses of NMDA, repeated electrical stimulation and indeed raisedextra cellular calcium levels, all result in an effect on calciumpotentials similar to that seen for AChE peptide. The most obviouscommon factor in these three other treatments is that all to themenhance calcium entry; the most parsimonious explanation for thereduction in calcium potential, seen following AChE peptide, is that thepeptide enhances calcium entry too.

These results suggest that the peptide specified in the invention isenhancing calcium entry into a population of neurons in the substantianigra. Once large amounts of calcium have entered the neuron, bufferingmechanisms come into play, reflected by the marked reduction in calciumpotential. At its most effective, when the peptide is combined with thefragment from β-amyloid, then this enhanced calcium entry followed bythe triggering of intracellular control mechanisms, is seen as aspontaneous oscillation. It has already been shown that recombinantAChE, acting in a non-classical fashion, can enhance calcium entry intothese neurons via a modulatory action on the NMDA receptor. Theseresults suggest that the peptide derived from AChE, specified in theinvention, could be responsible for this effect.

(ii) Behavioural Studies

In these experiments, rats were chronically implanted with a cannula inone substantia nigra and left to recover. After a period of about 3days, they were infused with either a saline control solution, or asolution containing the 14-mer AChE peptide of the invention at a doseof 1 μl of 10⁻⁵M. After a single infusion, they were challenged dailywith a systemic application of amphetamine for the subsequent 10 days.Although the control group (n=6) showed no significant effects, thegroup receiving the peptide (n=8) gradually started to displaycontraversive rotation, which reached a maximum after 7 days postinfusion and remained consistent for the remaining 3 days tested.

These results suggest that the peptide-mediated calcium entry observedin (i) could be setting in train long-latency, long-term intracellularevents that result in a sustained elevation of the activity of neuronsin the treated substantia nigra. This enhanced, unilateral activation ismanifest as contraversive circling behaviour.

AChE when infused unilaterally into the substantia nigra produces along-term increase in circling behaviour which reflects increasedactivity of the nigrostriatal pathway. Under some circumstance thiseffect is mimicked by AChE-peptide, although the onset of the effecttakes several days following peptide infusion and the response isvariable. A low concentration of APP-peptide also increases activity ofthe nigrostriatal pathway, although doubling the concentration reversesthis effect, possibly reflecting the change from trophic to toxicactions of this agent. AChE- and APP-peptide appear to interact.

EXAMPLE 4 Electrophysiological Evidence for an Effect of the AChE 14-merPeptide on Neurones of the Hippocampus

The hippocampus is a brain region remote from the substantia nigradetailed in the original application. This issue is important because aneffect of the peptide in the substantia nigra can be connected toParkinson's disease because cells in the substantia nigra are lostduring the development of this condition. By contrast an effect in thehippocampus can be connected to Alzheimer's disease because thehippocampus is a major site of degenerative neuropathology inAlzheimer's disease.

Data has been obtained to suggest that the peptide has direct toxiceffects on hippocampal cells in organotypic cultures. The effect issynergistic with the known excito-toxic effects of N-methyl-D-aspartate(NMDA), can be seen within one hour of application. Toxic effects arealso detectable histochemically over a culture period of three weeks.This is particularly important because there is a need for ademonstration of peptide toxicity in a system related to a majorneurodegenerative disorder.

The organotype tissue culture technique requires postnatal (day 5–7)rats given terminal anaesthesia followed by decapitation. Sections ofhippocampus, 400 μm thick, are prepared and then plated on aplasma/thrombin clot. A serum-based media is added to these cultures,which can then be maintained at constant temperature (35° C.) for up to21 days. After each study, cultures are stained with trypan blue toassess cell viability, in addition, after every removal of serum media,cultures are assayed for lactate dehydrogenase (LDH—a solublecytoplasmic enzyme used as an index of cellular damage).

Semi-acute application of the AChE-peptide and/or N-methy-D-aspartate(NMDA) for 1 hour results in extensive cellular damage in variousregions of hippocampal sections, compared to control samples.

Following chronic studies (cultures are maintained for 21 days andtreated with AChE-peptide and/or NMDA every 3–4 days), cultured cellsare immunocytochemically stained for acetylcholinesterase in order toassess what action, if any, the AChE-peptide has on cell number. Thefindings of biochemical studies (LDH assays) carried out on thesechronic cultures support the proposed toxic action of AChE-peptidefollowing its semi-acute application to hippocampal sections. However,current work suggests that acetylcholinesterase-positive cells may beprotected to some extent and, therefore it is possible that eitheracetylcholinesterase-negative cells are selectively vulnerable or theAChE-peptide may have a more pronounced action when applied with NMDA orboth these cases may apply.

EXAMPLE 5 Reproducible Binding of Peptide to Brain Sections

In order to obtain a probe derived from the AChE-peptide that could befollowed when bound to specific sites within sections of brain (whetherrodent or human) a modified peptide was made. The modified peptideconsisted of the AChE-peptide to which was covalently attached at the Nterminus a fluorescein group. This modified peptide was bound to fixedpermeabilised brain sections, the excess unbound material washed off andthen the fluorescein group detected with an alkaline phosphataseconjugated monoclonal anti-fluorescein reagent. This in turn was washedto remove the unbound excess, and a substrate that produces a highlylocalised, coloured, insoluble precipitate in the presence of alkalinephosphatase was added in a solution of pH 8.4. Finally, the reaction wasstopped by lowering the pH to neutral (pH 7.0) and the distribution ofthe reaction product was examined by microscopy and photography.

This indirect ligand overlay method provides a significant amplificationof the weak signal due to peptide binding and overcomes a generalproblem of background due to non-specific sticking of first or secondreagents. A control with the modified peptide omitted was alwaysincluded, and a drug, levamisole, was routinely used to block theactivity of alkaline phosphatase enzymes that are present naturally inthe tissue. The result is a clean assay that shows peptide-dependentbinding reactions confined to certain brain regions and certainpopulations of cells within those regions.

The specific example of the cerebellum is a good one; the peptidespecifically labels a population of cells with neuronal morphologywithin the granular cell layer of the cerebellum in rats, guinea pigsand humans. The cells labelled represent a subpopulation of the cellspresent in this highly cellular layer. In rodents the staining ispresent in cell bodies, but excluded from the nucleus, and extends intolong processes, some of which extend into the molecular cell layer,forming a net around the Purkinje cells, which are themselves negative.

EXAMPLE 6 AChE 14-mer Peptide

Rabbits were immunised by conventional protocols with adjuvantcontaining a modified AChE-peptide. In this case, the modification wasto construct a covalent cluster of four copies of the AChE-peptideconnected via three lysine residues. This structure is known as amulti-antennary peptide, or MAP-peptide, and is known to give rise tomore potent stimulation of the recipient's immune response in manycases. The rabbits were repeatedly immunised and test bleeds were usedto follow the development of a response to the antigen.

The resulting antiserum is a high titre polycolonal antiserum withmarked specificity for the AChE-peptide; although the MAP-AChE peptideis recognised with the highest affinity, the reagent is still a potentfor binding monomeric AChE-peptide alone. The anti-AChE reactivity is ofthe IgG subclass, indicating that a secondary response has occurred inthe animals.

A skilled reader knows that a similar immunisation protocol in mice(giving the MAP-peptide in an adjuvant into the peritoneal cavity on anumber of occasions) would give rise to an immune response to the AChEpeptide in mice. This immune response could be immortalised by fusion ofimmunocompetent cells from the immunised mice (conventionally the spleenbut in principle lymph node also) with a nonsecreting myeloma cell lineusing fusogens such as polyethylene glycol or electrical discharges.Immortalised cell lines producing a reactivity of interest are derivedclonally by screening assays based upon the solid phase binding assayused to study the binding of the polyclonal antibody to the peptide. Thecells of interest are subcloned to purity, so their product is a singleimmunoglobulin species and the resulting pure hybridoma population ispreserved by freezing in liquid nitrogen and used to produce themonoclonal antibody that is then used in all of the ways that a skilledreader knows of for such reagents.

It is routinely possible to obtain the sequence of the variable regionsof the immunoglobulins produced by hybridomas selected in this way. Thiswould be done (as in previously published protocols for other unrelatedmonoclonal antibodies) by using the polymerase chain reaction to amplifyand clone the variable region sequences from messenger RNA isolated fromthe hybridomas of interest. These regions are then cloned back into arecombinant background that permits the engineering of their associationwith specific detecting enzymes or prosthetic groups for detection orpurification. The recombinant protein is produced in bacteria, or ininsect cells using the baculovirus expression system, or in mammaliancell lines.

EXAMPLE 7 Competition Assay

In order to be sure that the antibody recognition of peptides occurswithout the possible distortion due to the binding of the peptide to acharged plastic surface, an assay was used in which the criticalinteraction is studied in solution. Briefly, a solid phase assay was setup using the AChE-peptide on a plastic surface in a small well and anamount of the antibody which will give a large signal of known size whenassayed in this solid phase assay. An aliquot of the antibody waspreincubated with potential competitors for binding to the AChE-peptidebefore presenting the resulting mixture to the peptide bound to thewell. If the competitor has bound to the anti-peptide antibody duringthe preincubation, then there will be less free antibody available tobind to the immobilised peptide in the well, and the signal thatrecorded in the solid phase assay will be reduced.

This assay was first validated by demonstrating that the AChE-peptideitself could compete in this assay in a dose-dependent manner, and wasthen used to show that the antiserum generated is specific forAChE-peptide. This latter conclusion is drawn from the fact that evenexcessive doses of the APP or BuChE peptide could not compete for thebinding of antibody to immobilised AChE-peptide.

When human CSF was used as the competitor during the preincubation, highlevels of competition were recorded showing that human CSF contains acomponent that is structurally similar to the epitope(s) present in theAChE-peptide.

The Western blotting assay in which the proteins of the CSF areelectrophoretically separated according to size and then transferred toa nitrocellulose substrate addresses the question of the identity of thespecies in human CSF that is recognised by the anti-peptide antiserum.The array of proteins on the nitrocellulose sheet (or blot) is probedwith the anti-peptide antibody followed by a secondary antibodycovalently attached to alkaline phosphatase. When this assay isperformed the anti AChE-peptide antiserum is found to specificallydecorate a protein component at approximately 25,000 Dalton molecularmass. This is not the size of the AChE. The protein is present in allCSF samples from both normal and Alzheimer's disease patients.

When the complex antiserum is affinity purified using the 25,000 Daltonprotein as the ligand, the resulting antibody recognises the 25,000Dalton protein as expected, but also a second, apparently less abundant,protein. This second reactivity has the interesting and potentiallyimportant property that the size of the protein is smaller in Alzheimerdisease patients than in normal controls. Although the number of samplesso far looked at is small (3 AD and 3 normal), the effect is completelyconsistent, so perhaps this is a reproducible difference between the CSFof normal and AD patients. If this is confirmed, the potential for adiagnostic test is clear.

Peptide Length

The inventors have so far only studied one AChE 14-mer peptide length;the evidence that polypeptides of other sizes may contain the functionalregion is that the anti-peptide antiserum recognises a species of 25,000Daltons (much larger than the c2,000 Daltons of the AChE-peptide).Furthermore recombinant AChE that contains the part of the proteinencoded by exon 6 is an effective competitor in the above competitionassay, showing that the region is still recognised by the antibody whenit is attached to the parent protein.

Thus, the peptide used may not be uniquely functional because of itssize; the functional structure is encoded within the 14-mer, and can bedetected by antibody when present in the context of a much largerpolypeptide.

1. An isolated peptide consisting of a peptide of SEQ ID No. 1, saidpeptide having a calcium channel modulatory function.
 2. A probeconsisting of the peptide of claim 1, labeled with a signal moiety, orimmobilized on a solid support.
 3. The peptide as claimed in claim 1,which peptide is a fragment of acetylcholinesterase.
 4. The peptide asclaimed in claim 1, which peptide has been chemically synthesized.
 5. Amethod for obtaining an antibody comprising administering the peptideaccording to any one of claims 1, 3, and 4 as an antigen to obtain saidantibody.